Biomedical subjects

E R Simpson

Publications and source records attributed to E R Simpson.

At least 109 records · Page 6Linked to original sources

PubMed1993

cAMP-dependent and tissue-specific expression of genes encoding steroidogenic enzymes in bovine luteal and granulosa cells in primary culture.

Steroidogenic enzymes are differentially expressed throughout the ovarian cycle. The complex pattern of cell-specific up- and down-regulation accounts, at least in part, for the cyclic production of estrogens, androgens and progesterone. The gonadotropins follicle-stimulating hormone and luteinizing hormone are the main regulators of ovarian steroid hormone production and act primarily via the cAMP second-messenger system. Previous studies have identified cAMP-responsive sequences (CRS) in a number of genes encoding steroidogenic enzymes. In the present study we attempted to compare the cAMP responsiveness of some of these sequences with each other and with the classical cAMP-response element (CRE), as identified in the somatostatin gene. In addition, we were interested to determine whether or not the information for tissue-specific expression is contained by these sequences. Using transient transfection of reporter gene constructs, comprising the CRS of bCYP11A, bCYP17, hCYP21B and bovine adrenodoxin, we investigated cAMP-dependent and tissue-specific expression in primary cultures of bovine luteal and granulosa cells. Treatment of transfected luteal cells with forskolin markedly increased the expression of all but the CYP17-specific reporter gene constructs. A similar pattern of forskolin responsiveness was observed when these reporter gene constructs were transfected in bovine granulosa cells in primary culture. Furthermore, when a reporter gene construct containing the classical CRE genomic was transfected in bovine luteal cells, its expression was also highly stimulated upon treatment with forskolin. Thus, the classical cAMP/CRE system appears to be functional in these cells. Northern blot analysis of primary cultures of bovine luteal and granulosa cells revealed that bCYP17 and bCYP21B are not expressed in control and forskolin-treated cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenodoxin↗

PubMed1993

Regulation of CYP11A gene expression in bovine ovarian granulosa cells in primary culture by cAMP and phorbol esters is conferred by a common cis-acting element.

Production and secretion of steroid hormones throughout the ovarian cycle occurs in a highly episodic and coordinated fashion that requires precise and finely tuned regulatory mechanisms. The regulation of ovarian steroidogenesis by the gonadotropin follicle stimulating hormone (FSH) and luteinizing hormone (LH) as well as by other factors occurs, at least in part, at the level of expression of the genes encoding steroidogenic enzymes. The present study is aimed at the elucidation of regulatory mechanisms by which cyclic adenosine monophosphate (cAMP) and protein kinase C regulate cytochrome P450scc (CYP11A) gene expression in bovine granulosa cells in primary culture. As a first step we characterized the bovine granulosa cell cultures with regard to regulation of P450scc activity and mRNA levels upon treatment with forskolin and/or the phorbol ester TPA. Forskolin, a potent stimulator of cAMP generation, increased both progesterone secretion and P450scc mRNA levels. In contrast, treatment with TPA alone decreased both basal progesterone production and P450scc mRNA accumulation. Co-treatment with forskolin and TPA decreased progesterone and P450scc mRNA levels as compared to forskolin treatment alone. The possibility that TPA interfered with the forskolin-stimulated cAMP production could be excluded because simultaneous treatment of granulosa cells with TPA and forskolin potentiated the formation of cAMP. In order to identify regulatory sequences within the 5' flanking region of the bovine CYP11A gene, chimeric DNA constructs comprizing regions of the CYP11A gene fused to a beta-globin-derived reporter gene were transfected into granulosa cells in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Tissue-specific promoters regulate aromatase cytochrome P450 expression.

In the human, estrogen biosynthesis occurs in several tissue sites, including ovary, placenta, adipose, and brain. Recent work from our laboratory has indicated that tissue-specific expression of aromatase cytochrome P450 (P450arom), the enzyme responsible for estrogen biosynthesis, is determined, in part, by the use of tissue-specific promoters. Thus the expression of P450arom in human ovary appears to utilize a promoter proximal to the translation start-site. This promoter is not utilized in placenta but instead, the promoter used to drive aromatase expression in placenta is at least 40 kb upstream from the translational start-site. In addition, there is a minor promoter used in the expression of a small proportion of placental transcripts which is 9 kb upstream from the start of translation. Transcripts from these promoters are also expressed in other fetal tissues including placenta-related cells such as JEG-3 choriocarcinoma cells, hydatidiform moles, and other fetal tissues such as fetal liver. On the other hand, in adipose tissue expression of P450arom may be achieved by yet another, adipose-specific promoter. The various 5'-untranslated exons unique for expression driven by each of these promoters are spliced into a common intron/exon boundary upstream from the translational start-site. This means that the protein expressed in each of the various tissue-specific sites of estrogen biosynthesis is identical.

Amino Acid Sequence↗

PubMed1993

Maintenance and regulation of 17 alpha-hydroxylase expression by bovine thecal cells in primary culture.

We have developed and characterized a primary cell culture system to study the regulation of 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) gene expression in bovine thecal cells. Conditions have been established for the dispersal and growth of thecal cells isolated from bovine follicles, which maintain the expression of P45017 alpha for up to 8 days. Bovine theca interna cells were grown to subconfluence and transferred into medium containing forskolin, a stimulator of adenylate cyclase. Levels of P45017 alpha transcripts reached a maximum value after 48 h of stimulation with forskolin. Added progesterone was converted to 17 alpha-hydroxyprogesterone at a rate of 214 pmol/mg protein.h in cells treated with forskolin for 72 h, whereas in control cells, the rate was 9.2 pmol/mg protein.h after 72 h. This was reflected in a 10-fold increase in endogenous androstenedione production by forskolin-stimulated cells. Studies employing various growth factors suggest that transforming growth factor-beta, but not basic fibroblast growth factor, is a potent inhibitor of forskolin-induced 17 alpha-hydroxylase activity and androstenedione production in these cells. We have also characterized this cell culture system with respect to expression of other steroidogenic enzymes. Cholesterol side-chain cleavage cytochrome P450 and 3 beta-hydroxysteroid dehydrogenase transcripts as well as endogenous progesterone accumulation were increased in response to forskolin stimulation. On the other hand, aromatase cytochrome P450 expression was undetectable. The ability to maintain bovine thecal cells, which retain 17 alpha-hydroxylase activity, in culture will provide a model system to study the regulation of expression of the P45017 alpha gene in the bovine ovary.

Animals↗

PubMed1993

Isolation and characterization of a complementary deoxyribonucleic acid insert encoding bovine aromatase cytochrome P450.

Aromatase, an enzyme complex comprised of aromatase cytochrome P450 (P450arom; the product of the CYP19 gene) and the flavoprotein NADPH-cytochrome P450 reductase, catalyzes the conversion of androgens to estrogens. Three cDNA inserts encoding P450arom were isolated from a bovine placental cDNA library. These inserts were sequenced and found to correspond closely to human P450arom sequence from the internal EcoRI restriction site (exon III) through the termination codon (exon X) into the 3'-untranslated region. The rapid amplification of cDNA ends technique was used to generate the rest of the cDNA 5' of the internal EcoRI site, using mRNA obtained from bovine granulosa cells as a template. This insert was sequenced, and when aligned with the other inserts, an open reading frame was found which was predicted to encode a protein of 503 amino acid residues. The deduced polypeptide shares 84% identity with human P450arom and 79%, 76%, 71%, and 57% identity with mouse, rat, chicken, and trout P450arom, respectively. A full-length open reading frame was generated using the polymerase chain reaction and mRNA obtained from bovine granulosa cells as template. After this insert was ligated into the pCMV5 expression vector, it was transfected into COS-1 monkey kidney tumor cells. We were able to demonstrate aromatase activity by assaying the incorporation of tritium into [3H] water from [1 beta-3H]androstenedione. Northern analysis revealed a single transcript of approximately 6 kilobases in poly(A)+ RNA obtained from bovine placental tissue and granulosa cells. This indicated for the first time a correspondence between the pattern of estrogen biosynthesis throughout the bovine ovarian cycle and the levels of transcripts encoding P450arom. In addition, weak hybridization was noted to transcripts of the expected size, namely 3.4 and 2.9 kilobases, in poly(A)+ RNA obtained from human placental tissue. The large size of the bovine transcript is due to a long 3'-untranslated region, because, based on the rapid amplification of cDNA ends technique, there appeared to be approximately 150 basepairs 5' of the start site of translation, and we were never able to find a polyadenylation site, even in one clone that went well past the corresponding polyadenylation site in human P450arom.

Amino Acid Sequence↗

PubMed1993

Polymerase chain reaction amplification fails to detect aromatase cytochrome P450 transcripts in normal human endometrium or decidua.

It has been proposed that the biosynthesis of estrogens by the human endometrium may be of physiological significance during the menstrual cycle. Local estrogen production was also suggested to be important in the development of endometrial cancer; however, the presence or absence of aromatase enzyme activity in normal human endometrium is controversial. To address this issue, we used a sensitive technique capable of detecting mRNA transcripts present in only very low copy number. The polymerase chain reaction linked to reverse transcription (RT-PCR) was used to evaluate the presence or absence of aromatase cytochrome P450 (P450arom) transcripts in endometrial tissues (n = 7) and endometrial stromal cells (n = 9) under various culture conditions. RNA was isolated from four proliferative and three secretory tissue samples and from cultured endometrial stromal cells isolated from seven proliferative and two secretory endometria. Five sets of cultures were treated with medroxyprogesterone acetate (MPA), estradiol (E2), and forskolin. Additionally, RNA was isolated from decidualized endometrium obtained from a patient with tubal pregnancy. A single stranded cDNA was synthesized from total RNA using Moloney murine leukemia virus reverse transcriptase and a P450arom-specific oligonucleotide. The single stranded cDNA was used as a template for PCR and was amplified for 20-35 cycles using P450arom-specific primers. RNA from adipose tissue and placenta was amplified to provide positive controls, whereas myometrial RNA was used as a negative control. In two experiments involving two endometrial tissues and three sets of cells in culture, a rat P450arom cRNA was coamplified in each sample as an internal control to demonstrate that the remote possibility of RT-PCR failures in individual test samples cannot account for our negative results. By Southern or slot blot hybridization of the amplified fragments using human and rat P450arom-specific probes, we found no evidence for the presence of P450arom transcripts in normal endometrium, decidualized endometrium, or endometrial stromal cells in culture. In our hands, assay of aromatase activity using [3H]water release from [3H]androstenedione by endometrial stromal cells in culture treated with MPA and E2, did not reveal any detectable aromatase activity. The same cells responded to MPA plus E2 treatment by a significant increase in PRL secretion into the culture medium. Presently, RT-PCR is the most sensitive method available for the detection of specific mRNA species in low copy numbers. These findings are indicative of the absence of P450arom transcripts in normal human endometrium.

Aromatase↗

PubMed1993

A link between breast cancer and local estrogen biosynthesis suggested by quantification of breast adipose tissue aromatase cytochrome P450 transcripts using competitive polymerase chain reaction after reverse transcription.

C19 steroids are converted to estrogens in a number of tissues by a specific form of cytochrome P450, namely aromatase cytochrome P450 (P450arom). Adipose tissue is the principal site of estrogen formation in postmenopausal women. Aromatase activity as well as P450arom transcripts primarily reside in the stromal cell component of the adipose tissue. Studies designed to investigate whether increased local aromatase activity in breast adipose tissue influences the growth of breast cancers have yielded discrepant results. In an attempt to clarify this controversy, adipose tissue was obtained from the four breast quadrants at the time of mastectomy (n = 13) performed for removal of a tumor. Breast fat P450arom messenger RNA levels were quantified and compared between the four quadrants within each specimen using competitive polymerase chain reaction after reverse transcription in which 10 micrograms human adipose total RNA together with 1 pg rat complementary RNA (internal standard) were reverse transcribed and coamplified. In 9 out of 13 patients (69%), highest P450arom transcript levels colocalized to the quadrants bearing tumors. This correlation was statistically significant (P < 0.001). The regional distribution of P450arom transcripts in breast adipose tissue of disease-free individuals, obtained during reduction mammoplasty (control group, n = 9), did not favor any particular region of the breast. We also quantified by morphometry the histological components of the adipose tissue samples from each quadrant in mastectomy specimens. The distribution of stromal cells significantly correlated with the distribution of P450arom transcript levels, in that quadrants containing highest proportions of stromal cells matched to highest transcript levels (P < 0.01). Although the quadrants bearing tumors contained the highest percentage of stromal cells, this correlation was not statistically significant. The adipose tissue surrounding a breast tumor displays increased estrogen biosynthesis, which may promote tumor growth. It is further suggested that the distribution of stromal cell components in breast adipose tissue gives rise to locally elevated P450arom expression, which in turn may favor neoplastic development and growth in these predisposed areas of the breast. The correlation between the presence of a tumor and elevated P450arom levels in the proximal adipose tissue is independent of tumor size, node involvement, histological type or grade, estrogen/progesterone receptor status, DNA index, or S-phase fraction.

Adipose Tissue↗

PubMed1993

Use of tissue-specific promoters in the regulation of aromatase cytochrome P450 gene expression in human testicular and ovarian sex cord tumors, as well as in normal fetal and adult gonads.

We have previously demonstrated that the tissue-specific regulation of human aromatase cytochrome P450 (P450arom) gene expression is, in part, the consequence of the use of tissue-specific promoters. Promoter I.1 (PI.1) and PI.2-specific transcripts are expressed in the placenta, whereas promoter II (PII) appears to be the only active promoter in the corpus luteum. Testicular and ovarian sex cord tumors with annular tubules (SCTATs) associated with gynecomastia in prepubertal boys and isosexual precocity in girls with Peutz-Jeghers syndrome (P-JS) have been previously reported. In the present study, we investigated the regulatory elements directing P450arom gene transcription in samples of SCTAT from three prepubertal boys and a girl with P-JS and an ovarian granulosa cell tumor from an adult woman, as well as in healthy fetal and adult testicular and ovarian tissues. Placental tissue was used as a control. Using polymerase chain reaction linked to reverse transcription and northern blotting, we determined the tissue-specific use of various P450arom promoters by analyzing specific 5'-termini from messenger RNA templates. Results indicate a universal gonadal promoter (PII) directs P450arom gene expression in healthy fetal and adult ovaries and testes, as well as in SCTAT of the P-JS and an adult ovarian granulosa cell tumor. These results are interpreted to mean that use of PII in human ovary and testis is preserved from the fetal period into adult life as well as in transformed neoplastic Sertoli and granulosa cells. On the other hand, transcripts from placenta are specific for PI.1 (and to a much lesser extent, PI.2). In SCTAT, immunoreactive P450arom is detected only in the cytoplasm of neoplastic cells, whereas the normal-appearing sex cords do not contain any immunoreactive P450arom. These results further suggest that the markedly increased aromatase expression of these transformed neoplastic cells is not a consequence of using different tissue-specific promoters. Rather it appears to involve activation (or failure of inhibition) of the upstream regulatory elements of the same promoter, which is normally functional in all gonadal tissues, namely the proximal PII.

Adolescent↗

PubMed1993

Functional domains of human aromatase cytochrome P450 characterized by linear alignment and site-directed mutagenesis.

The relationship of function to structure of aromatase cytochrome P450 (P450arom; the product of the CYP19 gene) has been examined by means of sequence alignment and site-directed mutagenesis. Comparison has been made between the sequence of P450arom and the two soluble bacterial cytochrome P450 isoforms, whose three-dimensional structure has been determined (P450BM3 and P450cam). From this comparison, it appears that although there is a similarity of overall structure in cytochromes P450, there is enough significant difference in the regions involved in substrate recognition and substrate binding that residues believed to be involved, even in the known structures, must be tested. With this in mind, we have generated a detailed alignment of P450arom, including the definition of putative alpha-helices and beta-sheets based on comparison of the alignments of P450BM3 and P450cam, generated from their three-dimensional structure, and have made mutations in regions we believe to be involved in substrate recognition at the solvent surface and orientation in the heme pocket. We have mutated F116 and F134 to determine if they are present in the heme pocket, and Q225 and L228 to determine if they are a part of the substrate recognition loop. Although F116E is essentially inactive and may be a folding mutant or may inhibit reductase binding, F134E is more active than the wild type and may be located in the heme pocket facilitating the hydrogen abstraction from C2 of androstenedione. Mutations at Q225 and L228 also result in the anticipated changes in the apparent Km and maximum velocity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Dexamethasone inhibits corticotropin-induced accumulation of CYP11A and CYP17 messenger RNAs in bovine adrenocortical cells.

The effect of dexamethasone on ACTH-induced accumulation of CYP11A and CYP17 mRNAs was studied in bovine adrenocortical cells in primary culture. The cells were treated with either ACTH (1 microM) or the adenylate cyclase activator forskolin (25 microM) and/or dexamethasone (100 nM). The accumulation of CYP11A and CYP17 mRNAs was evaluated by Northern blot analysis with the use of [alpha-32P]deoxy-CTP-labeled bovine CYP11A and CYP17 cDNAs. Chloramphenicol acetyltransferase (CAT) activity was monitored in bovine adrenocortical cells transfected with recombinant plasmids containing either CYP11A or CYP17 regulatory regions coupled to the CAT reporter gene and treated with forskolin and/or dexamethasone. Dexamethasone treatment of the cells cultured in the presence of ACTH or forskolin resulted in about 50% suppression of both CYP11A and CYP17 mRNA accumulation, with a concomitant fall in cortisol secretion to about 60% of the stimulated value. The effects of dexamethasone on accumulation of CYP11A and CYP17 mRNAs and cortisol secretion were blocked by pretreatment of the cells with RU 486 (100 nM), while RU 486 had no effect on forskolin-induced accumulation of either mRNA or cortisol secretion. Dexamethasone also inhibited the forskolin-induced expression of the transfected CYP11A- or CYP17-CAT constructs in bovine adrenocortical cells. The inhibitory effect of dexamethasone was greatly reduced by cotreatment of the transfected cells with RU 486. It is concluded that dexamethasone inhibits the ACTH-induced accumulation of CYP11A and CYP17 mRNAs at a transcriptional level and that the effect of dexamethasone is mediated by the glucocorticoid receptor.

Adrenal Cortex↗

PubMed1993

Tissue-specific promoters regulate aromatase cytochrome P450 expression.

In humans, estrogen biosynthesis occurs in several tissue sites, including ovary, placenta, adipose, and brain. Recent work from our laboratory indicates that tissue-specific expression of aromatase cytochrome P450 (P450arom), the enzyme responsible for estrogen biosynthesis, is determined, in part, by the use of tissue-specific promoters. Thus, the expression of P450arom in human ovary appears to utilize a promoter proximal to the translation start site. This promoter is not utilized in placenta; instead, the promoter used to drive aromatase expression in placenta is > or = 40 kb upstream from the translational start site. In addition, a minor promoter used in the expression of a small proportion of placental transcripts is 9 kb upstream from the start of translation. Transcripts from these promoters are also expressed in other fetal tissues, including placenta-related cells such as JEG-3 choriocarcinoma cells and hydatidiform moles and other fetal tissues such as fetal liver. In adipose tissue, on the other hand, expression of P450arom may be achieved by yet another, adipose-specific promoter. The various 5'-untranslated exons unique for expression driven by each of these promoters are spliced into a common intron/exon boundary upstream from the translational start site. This means that the protein expressed in each of the various tissue-specific sites of estrogen biosynthesis is identical.

Amino Acid Sequence↗

PubMed1992

Molecular basis of apparent isolated 17,20-lyase deficiency: compound heterozygous mutations in the C-terminal region (Arg(496)----Cys, Gln(461)----Stop) actually cause combined 17 alpha-hydroxylase/17,20-lyase deficiency.

The molecular defect in a reported case of isolated 17,20-lyase deficiency in a 46XY individual has been elucidated. The patient was found to be a compound heterozygote, carrying two different mutant alleles in the CYP17 gene. One allele contains a point mutation of arginine (CGC) to cysteine (TGC) at amino acid 496 in exon 8. The second allele contains a stop codon (TAG) in place of glutamine (CAG) at position 461 in exon 8 which is located 19 amino acids to the carboxy-terminal side of the P-450(17) alpha heme binding cysteine. COS-1 cells transfected with cDNAs containing one or the other of these mutations showed dramatically reduced 17 alpha-hydroxylase and 17,20-lyase activities relative to cells transfected with the wild type P-450(17) alpha cDNA. While the in vitro data in COS 1 cells can explain the patient's physical phenotype, with female external genitalia, it was somewhat discordant with the clinical expression of isolated 17,20-lyase deficiency with relative preservation of 17 alpha-hydroxylase activity in vivo. In addition to the expression studies of these two examples of mutants in the C-terminal region of cytochrome P-450(17) alpha, a third mutant cDNA construct containing a 4-base duplication at codon 480 previously found in patients with combined 17 alpha-hydroxylase/17,20-lyase deficiency was also expressed in COS-1 cells. This expressed protein was completely inactive with respect to both activities, supporting the biochemical findings in serum and in vitro biochemical data obtained using a testis from the patient. The results from these patients clearly indicate the importance of the C-terminal region of human P-450(17) alpha in its enzymatic activities.

Adolescent↗

PubMed1992

Canadian Mennonites and individuals residing in the Friesland region of The Netherlands share the same molecular basis of 17 alpha-hydroxylase deficiency.

A common mutation within the CYP17 gene that causes 17 alpha-hydroxylase deficiency, a form of congenital adrenal hyperplasia, has been found by direct sequencing of polymerase chain reaction (PCR) fragments of genomic DNA from six families residing in the Friesland region of the Netherlands. The mutation is a 4-base duplication within exon 8 of the CYP17 gene, which alters the reading frame encoding the C-terminal 26 amino acids of cytochrome P45017 alpha. This mutation has previously been found in two Canadian patients who are members of ostensibly unrelated Mennonite families. The Mennonite Churches derive their name from Menno Simons, an early leader of the sect in Friesland. Presumably this 4-base duplication appeared within the Friesian population prior to emigration of the Mennonites from the Netherlands.

Adrenal Hyperplasia, Congenital↗

PubMed1992

Brain aromatase cytochrome P-450 messenger RNA levels and enzyme activity during prenatal and perinatal development in the rat.

Aromatase cytochrome P-450 (P-450AROM) enzyme activity catalyzes the conversion of androgens to estrogens in specific brain areas. During development local estrogen formation is thought to influence the sexual differentiation of neural structures (i.e. increase neurite growth and establish neural circuitry) and modulate reproductive functions. This study was undertaken to investigate the ontogeny of the (P-450AROM) enzyme and its messenger RNA (mRNA) in medial basal hypothalamic (MBH) and preoptic area (POA) tissue during late fetal and perinatal development of the rat. Aromatase activity in the MBH-POA was negligible before gestational day (GD) 16 (< 0.1 pmol/h/mg protein), increased over 10-fold at GD 17 and continued to increase (over 5-fold) to peak levels at GD 19 (> 5.0 pmol/h/mg protein), and then declined to low levels at GD 22 and 2 days post-birth (approximately 1 pmol/h/mg protein). The profile of P-450AROM mRNA in the MBH-POA tissue was characterized by a predominant 2.7 kilobase (kb) mRNA species, similar in size to the largest functional P-450AROM mRNA observed in adult rat ovarian tissue. At GD 15, the P-450AROM mRNA was undetectable; low but detectable levels were seen at GD 17, the abundance increased at later time points and remained at peak levels on GDs 18 through 20, decreased slightly by GD 22, and then declined further by 2 days post-birth. The developmental increase in P-450AROM mRNA levels correlated with the ascending pattern of enzyme activity before GD 19, but the marked decrease in enzyme activity seen after GD 19 was not accompanied by a corresponding decline in mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Alternative promotion of aromatase P-450 expression in the human placenta.

We have previously reported the isolation and characterization of the human gene encoding aromatase cytochrome P-450 (P-450AROM). The gene had been demonstrated to span at least 52 kb and contain ten exons, the first of which, exon I.1, is untranslated. Here we report the isolation and characterization of a P-450AROM cDNA from a human placental primer-extended cDNA library which contains a unique 5' sequence. This cDNA has been isolated and sequences used to screen a human placental genomic library for the presence of a unique first exon. The exon (exon I.2) lies 9 kb 5' of the second, ATG-containing exon (exon II) and is spliced onto exon II at the same site as that reported for exon I.1. DNA sequence analysis indicates that exon I.2 has a putative TATA (TAAA) sequence 33 base pairs (bp) upstream from a putative transcription start site and putative CAAT (CATT) binding sequence beginning at 54 bp upstream from this start site. Polymerase chain reaction (PCR) amplification experiments indicate that mRNA containing exon I.2-specific sequences can be demonstrated in tissues of fetal, but not adult, origin. These data have been confirmed by Northern analysis in the placenta. Characterization of this genomic clone containing exons I.2 and II now establishes the P-450AROM gene to be at least 72 kb in length and raises new questions regarding tissue specific and developmental control of aromatase expression in the human.

Aromatase↗

PubMed1992

Ovarian aromatase cytochrome P-450 mRNA levels correlate with enzyme activity and serum estradiol levels in anestrous, pregnant and lactating rats.

We examined the changes in P-450AROM mRNA, aromatase enzyme activity and serum estradiol levels (E2) in anestrous, pregnant mare's serum gonadotropin (PMSG)-treated immature, pregnant, and lactating rats to determine if: (a) the various mRNA species encoding P-450AROM in rat ovarian tissue are differentially expressed during different hormonal states, and (b) a positive relationship exists between P-450AROM mRNA and enzymatic activity in rat ovarian tissue and serum estradiol levels from the same animals. Utilizing three different cDNAs encoding rat P-450AROM, levels of P-450AROM mRNA were determined by RNA blot analysis and scanning densitometry. Probe 1, a 5' probe, detects all three P-450AROM mRNA species in rat ovarian tissue (i.e. at 1.7, 2.2 and 2.7 kb). Probe 2 contains an unspliced intronic sequence in place of the heme-binding domain at its 3' terminus and thus the mRNA detected by this probe must encode a nonfunctional aromatase protein. Only the two smaller (i.e. nonfunctional) mRNA species at 1.7 and 2.2 kb are detected by probe 2. Probe 3 contains the heme-binding region and hybridizes to principally the largest mRNA transcript at 2.7 kb (but hybridizes also to a 1.7 kb mRNA transcript). Aromatase enzyme activity was measured by using a saturating concentration of [1 beta-3H]testosterone as substrate in the [3H]water-release assay while serum estradiol levels were determined by radioimmunoassay. In immature rats (IR) or lactating animals (LA) P-450AROM mRNA was not detectable along with low serum estradiol (IR approximately 2.8 pg/ml; LA approximately 0.2 pg/ml) and aromatase activity levels (IR approximately 0.8 pmol/h per mg protein; LA less than 0.5 pmol/h per mg protein). Anestrous animals treated with 5 IU of PMSG resulted in a clear increase (24 h later) in P-450AROM mRNA levels, in concert with a 4-fold increase in serum E2 (approximately 12.5 pg/ml) and aromatase activity (approximately 4.2 pmol/h per mg protein). During pregnancy, all three mRNA species were clearly detectable, but low serum E2 levels (approximately 0.6 pmol/ml) and P-450AROM mRNA abundance were observed at 3 days of gestation (DG). Levels of all three P-450AROM mRNA species increased markedly at 15 and 18 DG; thereafter, the levels declined at 20 DG and further decreased at 22 DG. However, regardless of the probe utilized (probe 1, 2 or 3) in the RNA blot analyses, the mRNA transcripts detected by each probe were expressed in a concerted fashion with respect to abundance and pattern.(ABSTRACT TRUNCATED AT 400 WORDS)

Anestrus↗

PubMed1992

Inverse relationship between ovarian aromatase cytochrome P450 and 5 alpha-reductase enzyme activities and mRNA levels during the estrous cycle in the rat.

In the present study, we examined the changes in enzyme activity and mRNA levels of aromatase cytochrome P450 (P450AROM) and 5 alpha-reductase in ovarian tissue from adult cyclic rats. For each stage of the estrous cycle, the enzymatic activities were quantified by means of the 3H2O-release assay in the case of P450AROM and thin-layer chromatography in the case of 5 alpha-reductase. Levels of mRNA encoding P450AROM and 5 alpha-reductase in the ovary were determined by Northern blot analysis utilizing 32P-labeled rat cDNAs as probes. Serum LH levels were determined by RIA. Three P450AROM mRNA species were detected (at 1.7, 2.2 and 2.7 kb) in ovarian tissue from cyclic rats. All three P450AROM transcripts were expressed in a co-ordinated fashion throughout the cycle. The P450AROM levels were highest during diestrus and proestrus, decreased during estrus while at metestrus the levels were nearly nondetectable. Conversely, one 5 alpha-reductase mRNA species at 2.5 kb was detected in ovarian tissue from cyclic animals. Ovarian 5 alpha-reductase mRNA levels were lowest during diestrus and proestrus, increased at estrus and were most abundant during metestrus; a pattern opposite to that of P450AROM. The pattern of change in P450AROM and 5 alpha-reductase activities paralleled that of the respective mRNA profiles but lagged behind the mRNA profiles by about 24 h, or one stage of the estrous cycle. Aromatase activity was 1.5 pmol/h/mg protein during diestrus, increased over 3-fold at proestrus (approximately 5.5 pmol/h/mg protein), decreased at estrus and declined to the lowest values at metestrus (approximately 1.0 pmol/h/mg protein). In contrast, the 5 alpha-reductase activity pattern was essentially the mirror image of the P450AROM activity pattern during the estrous cycle. 5 alpha-Reductase levels were lowest during proestrus (approximately 5 pmol/h/mg protein) and estrus (approximately 8 pmol/h/mg protein), increased over 3-fold during metestrus, while the highest activity levels occurred during diestrus (approximately 36 pmol/h/mg protein). The normalization of the P450AROM and 5 alpha-reductase mRNA levels and their respective enzyme activities revealed a correspondence between mRNA abundance and subsequent increases (24 h later) in enzyme activity levels during the estrous cycle. These findings suggest that: (a) a temporal relationship exists between the profiles of the enzymatic activities that follows the changes in the levels of their respective mRNAs and (b) an inverse pattern exists between P450AROM and 5 alpha-reductase in terms of both enzymatic activity and mRNA expression during the estrous cycle in rat.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

PubMed1992

Compound heterozygous mutations (Arg 239----stop, Pro 342----Thr) in the CYP17 (P45017 alpha) gene lead to ambiguous external genitalia in a male patient with partial combined 17 alpha-hydroxylase/17,20-lyase deficiency.

17 alpha-Hydroxylase deficiency is characterized by defects in either or both the 17 alpha-hydroxylase/17,20-lyase activities. We have, for the first time, elucidated the molecular basis of the deficiency in a male pseudohermaphrodite with ambiguous external genitalia resulting from partial combined deficiency of both activities. The patient is found to be a compound heterozygote, carrying two different inherited mutant alleles in the cytochrome P45017 alpha (CYP17) gene. One allele, from his mother, contains a stop codon (TGA) in place of arginine (CGA) at amino acid position 239 in exon 4. Because this occurs at the N-terminal side of the heme binding sequence, the putative resultant truncated protein is nonfunctional. The second allele, from his father, contains a missense mutation encoding the substitution of proline (CCA) by threonine (ACA) at position 342 in exon 6. Reconstruction of this mutation by site-directed mutagenesis into human P45017 alpha cDNA followed by expression in COS 1 cells leads to the same amount of immunodetectable P45017 alpha protein as found with expression of the normal P45017 alpha cDNA, although both the 17 alpha-hydroxylase and 17,20-lyase activities are found to be reduced to 40-45% of those of the normal enzyme. The presence of ambiguous external genitalia in this 46 XY individual indicates that greater than 20% of the total normal 17,20-lyase activity is required for complete virilization in the male.

Adrenal Hyperplasia, Congenital↗